Summary neuropathy that can result in amputations, and kidney disease

2472 PRIMER
Development 140, 2472-2483 (2013) doi:10.1242/dev.093187
© 2013. Published by The Company of Biologists Ltd
How to make a functional β-cell
Summary
Insulin-secreting pancreatic β-cells are essential regulators of
mammalian metabolism. The absence of functional β-cells leads
to hyperglycemia and diabetes, making patients dependent on
exogenously supplied insulin. Recent insights into β-cell
development, combined with the discovery of pluripotent stem
cells, have led to an unprecedented opportunity to generate
new β-cells for transplantation therapy and drug screening.
Progress has also been made in converting terminally
differentiated cell types into β-cells using transcriptional
regulators identified as key players in normal development, and
in identifying conditions that induce β-cell replication in vivo and
in vitro. Here, we summarize what is currently known about how
these strategies could be utilized to generate new β-cells and
highlight how further study into the mechanisms governing later
stages of differentiation and the acquisition of functional
capabilities could inform this effort.
Key words: β cell, Diabetes mellitus, Mammalian metabolism
Introduction
Diabetes mellitus is a metabolic disease that results from a failure
in glucose regulation, causing severe hyperglycemia, tissue/organ
damage and increased morbidity and mortality. Pancreatic β-cells
respond to high blood glucose levels by secreting the peptide
hormone insulin, which acts on other tissues to promote glucose
uptake from the blood, for example in the liver where it promotes
energy storage by glycogen synthesis (Powers and D’Alessio,
2011). The Centers of Disease Control (CDC) estimated that 25.8
million Americans had diabetes in 2010, and more than 300 million
people are affected worldwide according to the International
Diabetes Federation, thus making diabetes a major worldwide
healthcare challenge.
Diabetes is classified into two related but distinct diseases with
different causes. Type 1 diabetes results from autoimmune
destruction of insulin-producing β-cells in the pancreas. Type 2
diabetes, which is commonly associated with obesity, occurs when
insulin demand due to persistently high blood sugar overwhelms
the capacity of β-cells to produce sufficient insulin to prevent
hyperglycemia. In Type 2 diabetes, peripheral tissues, such as fat
and muscle, also become resistant to the effects of insulin. This
high demand on β-cells frequently leads to β-cell malfunction, dedifferentiation and death (Ashcroft and Rorsman, 2012; Talchai et
al., 2012). The number of β-cells lost in Type 2 diabetes is unclear
but can approach 60% (Butler et al., 2003; Rahier et al., 2008), and
the remaining β-cells are likely to be in some way dysfunctional.
In diabetes, the persistent misregulation of glucose homeostasis
also leads to a variety of secondary complications including
cardiovascular disease, retinopathy and associated blindness,
1
Department of Stem Cell and Regenerative Biology and 2Harvard Stem Cell
Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
*Author for correspondence (dmelton@harvard.edu)
neuropathy that can result in amputations, and kidney disease
leading to renal failure (Powers and D’Alessio, 2011). According
to the CDC, diabetes is the leading cause of kidney failure,
blindness and amputations in American adults. Cardiovascular
complications, which are even more common, lead to greatly
increased healthcare costs and reduced life expectancy (Caro et al.,
2002). Improving glycemic control could thus prevent these
complications and result in improved patient health (Fonseca,
2003).
A number of drugs exist to improve glycemic control and treat
diabetes, including administration of insulin itself. Treatment of
Type 1 diabetes requires continuous administration of exogenous
insulin, whereas Type 2 diabetes can often be controlled by other
oral or injected therapeutics that act on the β-cells or peripheral
tissues. However, none of these therapies matches the precision of
endogenous β-cells, and all have side effects including risk of
ketosis and coma (Nathan et al., 2009). For Type 1 diabetes, insulin
is the only option, and multiple blood glucose tests and insulin
doses per day, every day for the patient’s entire life, are required.
Alternative therapeutic options are crucial in order to address
these healthcare challenges and the field of regenerative medicine
is poised to contribute. Strategies that induce replication and
regeneration of existing β-cells could enhance the number of βcells available to control blood glucose, and studies of β-cell
replication in a variety of genetic models have identified candidate
pathways. In addition, the discovery of pluripotent embryonic stem
cells (ESCs) capable of developing into any cell type has inspired
a more radical strategy in which faulty or missing tissues are
completely replaced. Precedents for this approach have been set by
studies for other tissues, including ESC-derived cardiomyocytes
that engraft into injured heart muscle and prevent arrhythmias
(Shiba et al., 2012) and ESC-derived oligodendrocyte progenitor
cells that restore mobility in rats that have suffered spinal cord
injuries (Keirstead et al., 2005). β-cells make an especially
attractive case for cell replacement strategies because only a single
cell type is missing and replacement can occur in non-endogenous
sites, a surgical advantage as cells can be placed subcutaneously in
minimally invasive surgeries. Transplantation into a nonendogenous site also offers the opportunity to protect the
replacement cells from autoimmune attack because the replacement
cells can be transplanted inside immunoprotective devices,
enabling allogeneic replacement strategies as well as protected
autologous transplant for Type 1 diabetics. The transplantation of
pancreatic islets into the hepatic portal vein has already been
demonstrated as a very effective treatment for diabetes (Lacy and
Scharp, 1986; Mullen et al., 1977; Bellin et al., 2012; Shapiro et
al., 2000; Shapiro et al., 2006). However, the demand for human
cadaveric pancreata, from which the islets are isolated, far outstrips
the supply, especially as single patients often require more than one
donor. These results firmly establish the clinical value of generating
β-cells from alternative sources. In addition to their clinical value,
a reliable and reproducible source of human β-cells would be of
great benefit for in vitro studies of metabolism and β-cell function.
The development of new sources of human β-cells would also
DEVELOPMENT
Felicia W. Pagliuca1,2 and Douglas A. Melton1,2,*
Development 140 (12)
PRIMER 2473
ESC or iPSC
A. Directed
differentiation
Oct4
Nanog
Sox17
FoxA2
Pdx1
Nkx6.1
vit
ro
α-cells
Ngn3
dif
fer
ent
iatio
n
in
iv
in v
o
C. Replication
• Pax 4
• Extreme
injury
β-cell
B. Reprogramming
Acinar
• Youth
• β-cell loss
• Insulin resistance
• Systemic factors
• Betatrophin
• Pdx1
Ngn3
Mafa
Hepatocytes
Fig. 1. Strategies to generate new β-cells.
(A) Directed differentiation using growth
factors and small molecules can direct a
pluripotent stem cell (red) through the stages
of pancreatic differentiation in a manner that
mimics normal development. Currently,
functional β-cells can only be differentiated
through an in vivo transplantation step, but
deriving a bona fide β-cell fully in vitro
(dashed line) is a major goal. A subset of
important genes expressed at each stage is
listed. (B) Reprogramming of terminally
differentiated cell types, such as acinar or αcells, can be used to generate β-cells in vivo,
using the overexpression or injury strategies
listed. Reprogramming other mature cell
types, such as hepatocytes, fibroblasts or
neurons, in vitro into β-cells (dashed line)
remains to be achieved. (C) Inducing the
replication of existing β-cells is the primary
strategy for generating new endogenous βcells. Replication may be recapitulated in vitro
or induced in vivo with new small molecules
or proteins based on the strategies listed.
Fibroblasts
Neurons
Generating β-cells from pluripotent stem cells
Type 1 diabetics lack a sufficient number of β-cells and many
patients appear to have none. In Type 2 patients, β-cell mass is
also insufficient to maintain glycemic control. Therefore,
strategies to create new β-cells for therapeutic replacement have
garnered significant excitement in the last two decades. A major
advance toward this goal was the identification of pluripotent
human ESCs (hESCs) that are capable of generating tissues from
all three developmental germ layers (Thomson et al., 1998). In
the decade following this discovery, an additional source of
pluripotent stem cells was identified – induced pluripotent stem
cells (iPSCs) reprogrammed from murine fibroblasts (Takahashi
and Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007). Soon
thereafter, iPSCs were also engineered from human cells (Lowry
et al., 2008; Nakagawa et al., 2008; Takahashi et al., 2007; Yu et
al., 2007). One of the remarkable features of iPSCs is that, like
ESCs, they have the capacity to generate all cell types (Okita et
al., 2007; Wernig et al., 2007). Thus, these cells present an
unprecedented opportunity to generate replacement tissues in
vitro, including autologous cells from patient-specific cells. To
this aim, autologous mouse iPSCs were differentiated into
hematopoietic progenitors that were shown to be effective in
treating sickle cell defects in an anemia mouse model (Hanna et
al., 2007).
Similar disease-curing strategies through the directed
differentiation of pluripotent stem cells (Fig. 1A) should be possible
for diabetes, although one major hurdle exists: discovering strategies
that can recapitulate the development of functional β-cells
(summarized in Fig. 2) in vitro. During development, cells of the
fertilized embryo first select which germ layer fate to acquire
(ectoderm, mesoderm or endoderm); β-cells derive from the
endodermal layer. After endodermal specification, signals from
adjacent developing tissues induce specification of pancreatic
progenitors that have the potential to generate all three pancreatic cell
types: ductal, acinar and endocrine (Fig. 2). The exocrine tissue of
the pancreas is composed of ductal and acinar cells, whereas islets
provide the endocrine function of the pancreas. After selection of
endocrine fate, those endocrine progenitors must then be specified to
become one of the five endocrine cell types of the islet: insulinproducing β-cells, glucagon-producing α-cells, somatostatinproducing δ-cells, pancreatic polypeptide-producing (PP) cells, or
ghrelin-producing ε-cells (Fig. 2). A decade ago, even the first step
of this process – the controlled induction of endoderm – had not been
achieved from ESCs. In the intervening years, however, remarkable
progress has been made toward the ultimate aim of creating fully
functional β-cells from pluripotent cells in vitro.
Making definitive endoderm
Studies in mice, including important genetic models described
below, identified transcription factors that are key regulators of
pancreatic development. Sox17 and FoxA2 (Hnf3beta) are required
to generate endodermal tissue and the gut tube that derives from it
(Ang and Rossant, 1994; Kanai-Azuma et al., 2002; Weinstein et
al., 1994). FoxA2 acts in part through mediating nucleosome
depletion and subsequent gene activation (Li et al., 2012).
Molecular signals, in particular the TGFβ superfamily member
DEVELOPMENT
provide a potentially unlimited source of cells for the development
of a novel drug-screening platform for diabetes.
Here, we present an update on the progress in generating new βcells from three complementary strategies – pluripotent stem cell
differentiation, reprogramming from other cell types, and induction
of replication in existing β-cells (Fig. 1) – based on work in both
murine and human systems.
Development 140 (12)
2474 PRIMER
Inductive signals
Pdx1
Nkx6.1
Ptf1a
Sox9
Sox17
FoxA2
Pluripotent
Endoderm
Alb1
Cdx2
Pdx1
Nkx6.1
Ngn3
Neurod1
Pancreatic
endoderm
Sox9
Ptf1a
Ins
Endocrine
progenitor
β-cells
Gcg α-cells
Sst δ-cells
Mesoderm
Ectoderm
Duct
Liver
Intestine
Acinar
Ppy PP-cells
Ghrl ε-cells
Nodal, that induce embryonic differentiation into endoderm have
also been identified, using first frog and fish and later murine
models (Brennan et al., 2001; Tremblay, 2010). Another TGFβ
family member, activin, has similar receptor binding patterns as
Nodal, is easier to produce as a recombinant protein, and can signal
via similar downstream pathways (Chen et al., 2013). These
features of activin A enabled researchers to develop the first
efficient protocol for generating definitive endoderm in vitro from
pluripotent stem cells (D’Amour et al., 2005).
The transition to pancreatic endoderm
Definitive endoderm can subsequently differentiate into pancreatic
endoderm, provided that the appropriate cues are present. The key
pancreatic regulator Pdx1 (Ipf1) is expressed early in
embryogenesis downstream of FoxA1 and FoxA2, and marks the
region of endoderm committed to a pancreatic fate (Gao et al.,
2008; Ohlsson et al., 1993). In fact, deletion of this transcription
factor prevents pancreatic acinar and endocrine development
(Jonsson et al., 1994; Offield et al., 1996) and, based on lineagetracing experiments, all pancreatic cell types arise from Pdx1positive cells (Gannon et al., 2000; Gu et al., 2002). Additionally,
genetic analysis of patients suffering from a rare monogenic form
of diabetes called MODY (mature onset diabetes of the young)
confirms the importance of PDX1 and other transcription factors
in pancreatic differentiation, as causative mutations of these genes
have been identified in these patients (Ashcroft and Rorsman,
2012). For example, pancreatic agenesis is seen in patients
harboring certain homozygous point mutations in the PDX1 coding
sequence (Stoffers et al., 1997). The transcription factors Hnf1b
(Tcf2), Hnf6 (Onecut1) and Prox1 (Wandzioch and Zaret, 2009) are
also expressed in tissues from which the pancreas, as well as the
liver, is derived. In line with this, HNF1B, HNF4A and HNF1A
have all been identified as MODY genes and a cause of human
diabetes (Ashcroft and Rorsman, 2012). Thus, analysis of these
factors serves to guide whether stem cell differentiation in vitro is
recapitulating the essential gene expression patterns observed
during normal β-cell development (Fig. 2).
The pancreatic epithelium marked by Pdx1 expression can be
further subdivided into regions that will have different cell fates.
Of particular importance, pancreatic multipotent progenitors have
been identified in the tip and trunk regions of early branching
structures of the developing mouse pancreas (Schaffer et al., 2010;
Kopp et al., 2011a; Zhou et al., 2007). Based on lineage tracing of
carboxypeptidase A1 (Cpa1)-positive cells marked at E12.5 or
earlier, tip multipotent progenitors that co-express Cpa1, the
transcription factors Pdx1 and Ptf1a and high levels of cMyc
generate the three major cell types of the pancreas: endocrine,
acinar and ductal cells. Lineage tracing of Ptf1a-expressing cells
revealed similar results (Kawaguchi et al., 2002). Ptf1a itself is
required for the development of the exocrine pancreas, in particular
the acinar cells. In the absence of acinar cells, endocrine cells in
Ptf1a null mice develop but mismigrate to the splenic mesenchyme
(Krapp et al., 1998). After E12.5, the pancreatic epithelium
differentiates into at least two progenitor regions – the
Ptf1a+/Cpa1+ tip and the Sox9+/Hnf1b+/Nkx6.1+ trunk – that later
differentiate into acinar or ductal and endocrine cells, respectively
(Kopp et al., 2011b; Schaffer et al., 2010).
The development of pancreatic endoderm expressing these key
transcription factors is inhibited by sonic hedgehog (Shh) signaling
(Apelqvist et al., 1997). In vivo signals from the developing
notochord repress this signaling in the adjacent endoderm, allowing
it to differentiate into the pancreatic lineage (Hebrok et al., 1998;
Kim and Melton, 1998; Kim et al., 1997). Specification of both the
pancreatic and liver endoderm from the endodermal germ layer
also requires retinoic acid signaling (Chen et al., 2004; Martín et
al., 2005; Molotkov et al., 2005; Stafford and Prince, 2002).
Expansion and branching of this Pdx1-positive pancreatic
epithelium is promoted by Fgf10, which is presumably released by
the surrounding mesenchyme (Bhushan et al., 2001). Importantly,
the combination of these signals – retinoic acid, the Shh inhibitor
cyclopamine, and Fgf10 – is sufficient to induce pancreatic
epithelium in vitro from definitive endoderm derived from
pluripotent stem cells (D’Amour et al., 2006). For an extended
review of signaling during pancreatic differentiation see
DEVELOPMENT
Fig. 2. An overview of fate choices during normal β-cell development. Pluripotent cells first acquire the identity of one of three germ layers;
pancreatic cells arise from the endodermal layer. A subset of endoderm is specified by Pdx1 expression to become pancreatic endoderm, which will
subsequently differentiate to a pancreatic ductal, acinar or endocrine fate. Endocrine progenitors express Ngn3 and differentiate further into the five
hormone-expressing cell types of the islet according, at least in part, to which other transcription factors are expressed. A subset of relevant
transcription factors is listed. For a more extensive review, see Pan and Wright (Pan and Wright, 2011). Ins, insulin; Gcg, glucagon; Sst, somatostatin; Ppy,
pancreatic polypeptide; Ghrl, ghrelin.
McCracken and Wells (McCracken and Wells, 2012). Similar
protocols have been developed to enhance pancreatic progenitor
development and induce the expression of factors such as Nkx6.1
and Ptf1a, and these include the addition of bone morphogenetic
protein (BMP) inhibitors and protein kinase C (PKC) activators
(Chen et al., 2009; Kroon et al., 2008; Nostro et al., 2011; Rezania
et al., 2012).
Differentiation to endocrine cells
One of the more important advances in this field was the
demonstration that, when transplanted, the pancreatic progenitors
generated from two of these protocols can further differentiate into
functional, glucose-responsive β-cells in vivo after 4 months
(Kroon et al., 2008; Rezania et al., 2012). These in vivo
differentiated cells co-express insulin and key transcription factors,
including Pdx1, Nkx6.1 and Mafa, and are sufficient to rescue
diabetes in murine models, suggesting that they closely resemble
true β-cells. Although a remarkable advance, the generation of true
β-cells in vitro has not yet been achieved, and the mechanisms
through which differentiation from ESC-derived cells occurs in
vivo remain unknown. Furthermore, the number and identity of the
cells within the pancreatic progenitor population that are capable
of in vivo differentiation have not yet been established.
Past research has established that progenitors of the endocrine
lineage are pancreatic endoderm cells that transiently express the
transcription factor Ngn3 (Neurog3) (Gradwohl et al., 2000; Gu et
al., 2002). Lineage tracing has demonstrated that all islet endocrine
cell types derive from Ngn3-positive cells (Gu et al., 2002).
Therefore, the induction of NGN3 expression in hESC-derived
pancreatic progenitors is likely to be required to generate β-cells,
whether in vivo during transplantation or in vitro. Ngn3 expression
levels are regulated by Notch signaling, which represses Ngn3
expression and maintains the progenitor phenotype (Apelqvist et
al., 1999; Jensen et al., 2000). Directed differentiation protocols
using different combinations of Notch inhibitors, exendin-4, BMP
inhibitors, PKC activators, keratinocyte growth factor (KGF, or
Fgf7) or epidermal growth factor (EGF) have generated Ngn3positive cells in vitro after pancreatic epithelium induction
(D’Amour et al., 2006; Kroon et al., 2008; Rezania et al., 2012;
Schulz et al., 2012). Several days after the appearance of Ngn3positive cells, endocrine cells expressing insulin or glucagon (or
both) appear in these cultures. ESC-derived pancreatic progenitors
can also generate some Ngn3-positive cells (and subsequent
endocrine cells) without addition of any factors to the medium in
the last stage (Kroon et al., 2008). The most efficient current
protocols generate greater than 90% Pdx1-positive and greater than
60% Nkx6.1-positive pancreatic endoderm (Rezania et al., 2012;
Schulz et al., 2012). Nonetheless, despite variations in the protocol,
growth factors, small molecules and cell lines used, none of the
endocrine cells generated in vitro function as true β-cells.
Most importantly, ESC-derived insulin-expressing cells fail to
secrete insulin appropriately in response to the addition of various
concentrations of glucose. Normal islets or dispersed β-cells release
high levels of insulin in response to high levels of glucose in the
glucose-stimulated insulin secretion (GSIS) assay and can do so
repeatedly (Ashcroft and Rorsman, 2012) (Fig. 3). However, ESCderived β-cells fail to increase the amount of insulin secreted in
response to high versus low glucose (D’Amour et al., 2006). This
function is the key aspect of β-cell identity and the mechanism
through which β-cells control glucose metabolism in vivo.
Generating β-cells that can perform GSIS in vitro, as cadaveric
human islets can, is an important challenge for the field. Of note,
PRIMER 2475
Gadue and colleagues recently generated insulin-positive cells from
endodermal progenitor cell lines that secrete insulin, but it is not
yet clear how this insulin secretion compares to that obtained by
existing protocols or whether these cells co-express β-cell
transcription factors or function in vivo after transplantation (Cheng
et al., 2012). An analysis of young mouse islets of different ages
revealed the upregulation of some genes, including urocortin 3
(Ucn3), that correlates with increasing insulin secretion and might
be relevant to the acquisition of function in ESC-derived β-cells
(Blum et al., 2012; van der Meulen et al., 2012). In addition, the
insulin-expressing cells that have been generated from ESCs thus
far fail to express key β-cell-specific transcription factors, including
Pdx1, Nkx6.1 and Mafa, as well as other metabolic enzymes and
cell surface transporters related to normal β-cell function (Xie et
al., 2013). The reasons for this anomaly are not yet clear.
Following the fate of pancreatic progenitors
Lineage-tracing experiments that address how many of the
pancreatic progenitors go on to produce the functional graft have
not been performed, in part due to the challenges of hESC genomic
engineering. For example, only a subset of Pdx1- and Nkx6.1expressing cells may be competent to generate the graft of insulinexpressing cells (Fig. 4A), indicating the existence of an additional,
heterogeneously expressed factor. If only a few cells are competent,
this could explain why it takes 4 months for the transplanted graft
to become functional and able to control diabetes in the host
(Kroon et al., 2008; Rezania et al., 2012).
The analysis of an equivalent pancreatic progenitor stage during
human fetal development has also not been performed, and
therefore comparisons between endogenous human pancreatic
progenitors and those derived from stem cells have not been
possible. This type of analysis could determine the identity of
additional regulators that are normally present in this cell type but
are either not expressed in stem cell-derived progenitors or are
expressed in only a subset of cells.
Identifying signals for endocrine differentiation in vitro
If pancreatic progenitors are correctly programmed, a second
hypothesis to explain the lack of proper subsequent differentiation
in vitro is the presence of incorrect or incomplete signals to specify
β-cell fate. One strategy would be to identify small molecules or
culture conditions that promote the induction of endocrine fate at
the same time as promoting the expression of key transcription
factors such as Pdx1 and Nkx6.1. In addition, much focus has been
placed on screening for small molecules that modulate transcription
factor expression, but it might be equally relevant to screen for
factors that modulate the expression of Ucn3, metabolic enzymes
or membrane transporters such as glucokinase or glucose
transporters that might be misexpressed in ESC-derived endocrine
cells (Xie et al., 2013) (Fig. 4B).
Alternatively, functional β-cells could be generated from ESCderived pancreatic progenitors via viral-, plasmid- or RNA-based
expression of missing genes, in much the same way that functional
iPSCs have been generated (Okita et al., 2010; Takahashi and
Yamanaka, 2006; Warren et al., 2010). One prerequisite to this
approach is to know precisely which genes are misexpressed in in
vitro derived cells. To address this, a recent study by Sander and
colleagues analyzed gene expression and chromatin status along
the steps of directed differentiation in vitro as well as in functional
endocrine cells generated from in vivo engraftment (Xie et al.,
2013). Although a number of gene expression differences were
identified between in vitro and in vivo differentiated endocrine
DEVELOPMENT
Development 140 (12)
Development 140 (12)
2476 PRIMER
Islet
Pancreas
Depolarization
Glucose
K+
Sur1
Kir6.2
Ca2+
Glut2
Ca+ channel
ATP
K+
P
Ca2+
GCK
β-cell
Nucleus
Pdx1
Glis3
Foxp3
Eif2ak3
Ptf1a
Rfx6
Hnf1b
Hnf1a
Neurod1
Klf11
Pax4
Ca2+
Ca2+
Insulin
Insulin
granules
Blood vessel
cells, this analysis was performed on mixed populations of cells of
which only a minority were actually insulin-expressing endocrine
cells.
The exogenous expression of genes required for β-cell identity,
especially when combined with siRNA-mediated knockdown of
those that are overexpressed, might reveal the combination of
factors required for β-cell differentiation and function. In addition
to protein-coding genes, true β-cells also express non-coding
RNAs including microRNAs and long non-coding RNAs
(lncRNAs) (Lynn et al., 2007; Morán et al., 2012), which may
also modulate β-cell identity. For example, miR-375 has been
shown to inhibit GSIS, and deletion of the microRNA-processing
enzyme Dicer prevents β-cell differentiation in mouse knockout
models (Lynn et al., 2007; Melkman-Zehavi et al., 2011; Poy et
al., 2004). Thus, in addition to identifying the proteins that need
to be induced or repressed to confer true β-cell identity,
microRNAs might also need to be induced or exogenously
expressed before β-cell fate can be engineered from pancreatic
progenitors in vitro.
Maintaining β-cell identity and function during in vitro
culture
Finally, a third hypothesis to explain the lack of functional β-cells
found in ESC-derived cultures is that existing protocols are capable
of generating true β-cells but we simply lack the culture conditions
to maintain these cells in vitro or to train these new young β-cells
to respond to glucose appropriately. Human β-cells are generally
agreed to be a difficult cell type to keep alive and functional in
vitro. Even 48-72 hours of culture of human islets results in the loss
of half the islet cells, as well as dramatically decreased GSIS in
vitro and a reduced ability to restore normoglycemia after
transplantation into diabetic mice (Noguchi et al., 2012). These
data suggest that, even if true β-cells could be generated through
directed differentiation strategies, they might not survive and
function very well using current culture protocols. However,
several groups have reported the ability to maintain β-cells in vitro
for several weeks in certain culture conditions so that cells maintain
their ability to be functionally engrafted after transplantation
(Gaber et al., 2001; Keymeulen et al., 2006).
Unlike cell fate choices in which an inductive cue need only be
transitory, the maintenance of the terminal function of a cell in vitro
requires the maintenance of an environment that is sufficiently
similar to the in vivo context. When fully differentiated and
functional human islets are cultured, parts of the islet niche are
already present, including the extracellular matrix (ECM) and
adjacent endothelial and mesenchymal cells carried over from the
islet isolation. The addition of appropriate ECM proteins or other
cell types to ESC-derived insulin-positive cells may improve their
terminal differentiation and function in vitro. Human islets are
surrounded by a milieu of ECM and mesenchymal, endothelial,
DEVELOPMENT
Fig. 3. Functional β-cells respond to increasing glucose levels by increasing insulin secretion. In glucose-stimulated insulin secretion (GSIS),
glucose is transported into the cell via glucose transporters [e.g. Glut1 (Slc2a1) or Glut2 (Slc2a2), pink], where it is phosphorylated by glucokinase (GCK)
and converted into ATP by subsequent metabolic reactions. Rising ATP levels (e.g. rising ATP:ADP ratios) trigger the closure of potassium channels [Sur1
(Abcc8) and Kir6.2 (Kcnj11) subunits], membrane depolarization, and the opening of calcium channels (blue). The resultant rise in intracellular calcium
levels triggers the exocytosis of insulin-containing granules and hence leads to increased insulin levels in adjacent blood vessels. Human genetic
studies of maturity onset diabetes of the young (MODY) patients have identified a number of mutations that trigger diabetes, including those in genes
encoding transcription factors (depicted in the nucleus) and components of the GSIS pathway indicated in this figure.
Development 140 (12)
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PRIMER 2477
ESC
DE
Ins+, functional
PE
1
3
in vi
3
2
2
1
in v
i
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vo
1
1
1
1
tro
3
1
3
3
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Ngn3
B
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2
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Ins+, non-functional
Ins+, non-functional
ESC
DE
PE
t
en
Curr col s
o
t
o
r
p
Chemical screens
Endocrine
signals
96-well
plate
Ins+, functional
Co-culture
Genetic engineering
C
Ins+, non-functional
ESC
DE
PE
Ins+, non-functional
t
en
Curr r e
cultu
Chemical screens
96-well
plate
Improved
islet culture
Ins+, functional
Co-culture
Genetic engineering
neuronal and exocrine cells, many of which act to support β-cell
identity and function. Mesodermally derived aortic endothelial
cells, for example, help maintain Pdx1 expression (Lammert et al.,
2001) and induce Ptf1a expression (Yoshitomi and Zaret, 2004),
and mature islets secrete Vegfa to attract endothelial cells
(Nikolova et al., 2006). Organ-specific mesenchymal cell lines can
also induce replication in hESC-derived definitive endoderm
(Sneddon et al., 2012). Finally, it should be noted that bona fide βcells differentiate and function in a three-dimensional environment,
whereas the majority of research efforts are performed in twodimensional tissue culture.
Taken together, these observations suggest that modifications to
the culture conditions might improve functional β-cell generation
(Fig. 4C). Alternatively, the co-differentiation of whole islets or
islet organoids, rather than β-cells in isolation, might ultimately
prove a more successful approach to differentiating true β-cells.
Reprogramming other cell types into β-cells
An alternative strategy to differentiating β-cells in a stepwise
fashion from pluripotent stem cells is the reprogramming of
terminally differentiated cell types into β-cells. This type of direct
reprogramming has made it possible to generate iPSCs,
cardiomyocytes, hepatocytes and neurons from fibroblasts (Huang
et al., 2011; Ieda et al., 2010; Takahashi and Yamanaka, 2006;
Vierbuchen et al., 2010). Remarkably, each of these cases required
the overexpression of only two to four genes. In the last decade,
similar strategies have proven successful for reprogramming other
cell types into β-cells.
Acinar to β-cell reprogramming
One example of this reprogramming-based approach was the
demonstration of the direct conversion of mouse acinar cells to βcells in vivo via viral expression of particular genes (Zhou and
DEVELOPMENT
Fig. 4. Identifying how to improve directed differentiation into β-cells. (A) Lineage tracing of ESC-derived pancreatic endoderm could reveal how
heterogeneous this population is and which cells are competent to generate functional β-cells in vivo. Depicted is a hypothetical outcome whereby
pancreatic progenitor cell type 1, but not types 2 or 3, generates functional β-cells in the graft. Types 2 and 3 may turn on insulin expression in vitro but
may not be functional, and in vitro cues may be insufficient to direct further differentiation of the type 1 cells. (B) Chemical screening for novel small
molecules or growth factors, co-culture with instructive cell types or genetic engineering strategies might be necessary to trigger the in vitro
differentiation of pancreatic endoderm cells into functional endocrine cells that express the normal set of transcription factors. (C) Current islet culture
conditions are not optimal for inducing the function of the insulin-positive cells made in the dish. Thus, in vitro derived cells might not be instructed to
function in the absence of appropriate culture conditions. The identification of novel culture conditions will improve the chances of generating β-cells
in vitro. DE, definitive endoderm; PE, pancreatic endoderm.
Development 140 (12)
2478 PRIMER
α-cell to β-cell reprogramming
More recently, an additional example of adult cell reprogramming
to β-cells has been described from adult mouse α-cells. Mansouri
and colleagues discovered that the ectopic expression of Pax4 was
sufficient to convert α-cells into β-cells in vivo (Collombat et al.,
2009). Conversely, loss of Pax4 leads to loss of β-cells and a
concomitant increase in the number of α-cells (Sosa-Pineda et al.,
1997). These data reflect the shared developmental trajectory of
these two cells types and their similar gene expression programs.
Interestingly, conditions of near complete β-cell ablation may
trigger a similar sort of reprogramming in α-cells. Herrera and
colleagues created a transgenic mouse model that permits α-cell
lineage tracing and near total β-cell ablation using the diphtheria
toxin receptor system (Thorel et al., 2010). Nearly complete β-cell
destruction led to the eventual regeneration of β-cells but,
surprisingly, many of these cells derived from former α-cells.
Transgenically marked α-cells began producing insulin and to coexpress the adult β-cell markers Pdx1 and Nkx6.1.
The functional similarity and shared ancestry of these two cell
types might make their interconversion possible in extreme
circumstances, which in this case might be an extreme niche that
entirely lacks local insulin signaling. Recent studies revealed that
α-cells harbor bivalent chromatin signatures at genes that are active
in β-cells, such as Pdx1 and Mafa (Bramswig et al., 2013). A
bivalent chromatin signature contains both active and repressive
histone marks, suggesting that part of the reason that α-cells may
have the plasticity to be reprogrammed into β-cells is that β-cellspecific genes are already poised to be active. Treatment of islets
with a histone methyltransferase inhibitor resulted in Pdx1 and
insulin expression in glucagon-positive cells, suggesting the
potential for partial transcriptional reprogramming, a finding that
warrants further investigation (Bramswig et al., 2013).
Although it is not yet clear whether complete α-cell to β-cell
transdifferentiation can occur in humans, if appropriate
immunoregulatory conditions could be established in severe cases
of Type 1 diabetes it might be possible to mimic ‘extreme β-cell
loss’ and observe α-cell transdifferentiation. Alternatively, one may
hypothesize that the transplantation of hESC-derived α-cells (in the
absence of β-cells) should also lead to transdifferentiation of some
of those α-cells into β-cells in vivo. Finally, a clear demonstration
of this α-cell transdifferentiation in vitro, either by Pax4
overexpression or by some modulation of glucagon or insulin
levels in the culture niche, would provide stronger support for the
utility of this strategy. Given the newly established ability to
genetically manipulate human pluripotent stem cells (Ding et al.,
2013), one could lineage trace in vitro derived α-cells using the
glucagon promoter and follow their differentiation in culture or
after transplantation into mice.
Reprogramming from other cell types
A particular form of injury to the pancreas, partial duct ligation
(PDL), has been argued to increase islet mass, potentially via
transdifferentiation of adjacent tissues (Wang et al., 1995). In this
injury model, the pancreatic ductal cells proliferate while acinar
tissue is simultaneously lost. Based on these and other
observations, Xu and colleagues suggested that PDL can induce
Ngn3 re-expression and that the new adult endocrine progenitors
are able to differentiate into new β-cells (Xu et al., 2008). The
molecular triggers for this conversion and whether it can take place
in humans or in vitro remain unknown. This study also suggested
that it was ductal cells that turned on Ngn3 expression to
transdifferentiate into endocrine cells; however, ductal-specific
lineage tracing to test this hypothesis indicated that Sox9-positive
ductal cells are not a source of new β-cells after PDL or after β-cell
ablation (Kopp et al., 2011b; Solar et al., 2009). In addition, several
recent studies have demonstrated that total β-cell number may not
actually change after PDL and that the β-cell mass increase might
be an artifact of the estimation methodology (Chintinne et al.,
2012; Rankin et al., 2013). However, a new study using Ptf1a
lineage tracing demonstrated that Ptf1a-positive acinar cells can
transdifferentiate into Ngn3-positive progenitors after PDL and that
these cells subsequently differentiate into new endocrine cells at
very low frequency (Pan et al., 2013). The insulin-expressing cells
derived from the former Ptf1a-positive acinar cells in this model
express mature β-cell markers, including Pdx1, Nkx6.1 and Mafa,
similar to the β-cells derived from virally reprogrammed acinar
cells (Pan et al., 2013; Zhou and Melton, 2008).
These lineage-tracing studies performed after injury also provide
evidence for the limited role that β-cell neogenesis plays
postnatally in maintaining β-cell mass. The bulk of evidence to date
suggests that the pancreas has a limited capacity to regenerate, and
the reader is referred to other reviews for a more extensive
discussion of the contradictory studies on the potential of β-cell
neogenesis to contribute new β-cells (Collombat et al., 2010;
Desgraz et al., 2011).
DEVELOPMENT
Melton, 2008). This example showed that, from an initial test of
nine transcription factors expressed in β-cells and their immediate
precursors, three factors – Ngn3, Pdx1 and Mafa – were sufficient,
once transduced into acinar cells, to reprogram those cells to a βcell fate (Zhou and Melton, 2008). Lineage tracing in vivo
confirmed that Cpa1-positive mature acinar cells could convert into
insulin-expressing cells post-transduction. Importantly, these
insulin-expressing cells also co-expressed key markers of β-cell
function including Nkx6.1, Glut2 (Slc2a2) and glucokinase, but no
longer expressed key regulators of acinar function including Ptf1a
or amylase and did not express any of the other islet hormones.
Finally, these induced cells were sufficient to improve glycemic
control in diabetic mice, a key characteristic of bona fide β-cells
(Zhou and Melton, 2008).
Nonetheless, these factors are not universally sufficient to
generate β-cells; they are insufficient to reprogram skeletal muscle
or fibroblasts into β-cells. Although acinar cells have been
reprogrammed in vivo, no mouse or human cell type has been fully
reprogrammed into a β-cell in vitro. This might relate to the poorly
defined culture conditions required for the maintenance of β-cell
identity and function in vitro, as discussed above. Furthermore, the
reprogrammed acinar cells do not aggregate into islets, perhaps
owing to the lack of other reprogrammed islet cell types such as αcells, and hence these factors appear to be sufficient only to
generate β-cells. Most importantly in the context of potential
therapeutic application, this achievement will need to be
recapitulated either in vivo without viruses or, perhaps as a safer
option, in vitro for subsequent cell transplantation. The latter goal
would be particularly important to achieve from human cells, as it
is not clear whether the same factors used for the
transdifferentiation of mouse acinar cells will be sufficient in
another species. Reprogramming from other more easily accessible
and expandable cell types would also be advantageous. However,
directly reprogramming such cell types, which include fibroblasts,
hepatocytes and pluripotent stem cells, into functional β-cells might
require a different, or expanded, set of transcription factors together
with optimized culture conditions to provide the necessary niche.
The role that reprogramming will play in regenerative medicine
for the treatment of diabetes remains to be seen. Clearly, terminally
differentiated cells can be reprogrammed into other cell types by a
variety of overexpression strategies. These strategies for β-cells
have been performed exclusively in vivo thus far, and so a
demonstration of successful reprogramming in vitro would be a
significant advance. Successful reprogramming would need to be
validated by more than the simple detection of insulin-expressing
cells, as many of the reprogramming factors, such as Pdx1, can
bind and activate the insulin promoter directly (Ohlsson et al.,
1993). The induction of other canonical β-cell genes, such as Glut2
and Nkx6.1, in the ‘reprogrammed’ cell would provide stronger
evidence of cell fate change than the expression of insulin alone.
In addition, whole-genome expression profiling of reprogrammed
β-cells and comparison with endogenous β-cells can help to
address how close the reprogrammed cell is to a bona fide β-cell.
Establishing function by in vitro GSIS assays or transplantation and
regulation of host blood glucose levels can provide valuable
additional evidence as to how closely a reprogrammed cell
resembles a β-cell.
In addition, reprogramming from accessible tissues, including
direct reprogramming from ESCs or fibroblasts and particularly
human tissues, should be a near-term goal. Finally, the
misregulated insulin-expressing cells that are currently being
generated from pluripotent stem cells represent a potentially ideal
target for reprogramming, as much of the pancreatic program
should already be in place.
Regenerating β-cells by induced replication
Whereas β-cell neogenesis may be achieved from pluripotent stem
cells or via reprogramming, the strategy for generating more β-cells
endogenously is to induce replication from existing β-cells
(Fig. 1C). Whereas tissues such as the blood or skin are regenerated
via the differentiation of tissue-specific stem cells, new pancreatic
β-cells normally derive from the replication of existing β-cells (Dor
and Melton, 2004). The replication of endogenous β-cells would
provide an autologous source of new β-cells and potentially
decrease the burden on existing β-cells that are overworked in Type
2 diabetes. In addition, it should be noted that some Type 1 diabetes
patients retain some β-cells; in one study, 16% of patients had
residual β-cell function as measured by detectable C-peptide levels
(Schölin et al., 2004). C-peptide, or connecting peptide, is released
from proinsulin processing and secreted in an equimolar ratio with
insulin. The persistence of functional β-cells in this patient
subpopulation suggests that they might benefit from therapies that
induce replication of these residual cells, if this strategy can be
combined with therapies to control the autoimmune attack.
Despite these potential advantages, a particular risk of such a
replication induction strategy is the inadvertent promotion of
tumorigenesis. The risk of cancer would be particularly important
to consider if acinar or ductal tissue was induced to proliferate
alongside β-cells. Conversely, this risk would be ameliorated if the
replication-inducing agent of choice was developed to have a high
specificity for β-cells relative to other cell types.
Young β-cells have higher replication rates
The rate of β-cell replication drops precipitously during human
aging, from 3% replicating cells among fetal β-cells to less than
0.5% by 6 months of age, and even lower thereafter (Kassem et al.,
2000; Perl et al., 2010; Teta et al., 2005). In another study, 2.5% of
β-cells in young mice (5 weeks old) replicated, whereas the cells
of old mice replicated at a rate of only 0.2% (Stolovich-Rain et al.,
PRIMER 2479
2012). The rate of β-cell replication in a diphtheria toxin-based βcell-ablation model reaches 7.5% in young mice, but still reaches
as high as 1% in old mice (Stolovich-Rain et al., 2012), suggesting
that an intrinsic capacity to replicate is maintained in old age but
also that youth itself is one variable that promotes β-cell
replication. Whether this change in replication capacity is
autonomous or due to systemic ‘aging’ factors remains to be
determined.
The replication of β-cells also increases during pregnancy,
potentially via the activities of prolactin and placental lactogen,
although the increase in β-cell mass may be less in humans than in
rodents (Butler et al., 2010; Parsons et al., 1992; Van Assche et al.,
1978). Prolactin may act in part via repressing the transcriptional
regulator menin, a protein that when overexpressed can prevent
pregnancy-associated replication (Karnik et al., 2007).
Alternatively, pregnancy may primarily influence the amount of
insulin secretion rather than the number of β-cells (Brelje et al.,
2004).
Screening for small-molecule effectors that modulate
replication
In mice, treatment with exendin-4, a glucagon-like peptide 1
receptor (Glp1r) agonist, led to a threefold increase in BrdUpositive replicating β-cells (Xu et al., 1999; Stoffers et al., 2000).
Exendin-4 has a longer half-life than glucagon-like peptide 1 itself,
making it more clinically useful (Eng et al., 1992). The replicationinducing effects of exendin-4 have also been observed in young
(<22 years) human islets transplanted into mice but not in old (>35
years) human islets (Caballero et al., 2013; Tian et al., 2011).
Additional molecular regulators of β-cell replication in rodents
have been identified through high-throughput screening of
reversibly immortalized β-cell lines or primary rodent islets. These
regulators include phorbol esters, thiophene-pyrimidines,
dihydropyridine derivatives and adenosine kinase inhibitors (Annes
et al., 2012; Wang et al., 2009). Glucose or Glp1r agonists have an
additive effect on the replication induced by most of these recently
identified factors (Annes et al., 2012; Wang et al., 2009); however,
it remains to be seen whether these small molecules will have an
inductive effect on human islets. More recently, Schultz and
colleagues identified a novel small molecule, WS6, that increased
both rat and human β-cell replication in vitro by sixfold in
dispersed islets and by more than tenfold in intact islets (Shen et
al., 2013).
Identifying mechanisms of replication induction using the
LIRKO model
Given the role of insulin signaling in the development of diabetes,
liver-specific insulin receptor knockout (LIRKO) mice were
generated to investigate the role of insulin signaling in hepatocytes.
Surprisingly, mutation of the insulin receptor in hepatocytes
resulted not only in insulin resistance and glucose intolerance but
also led to a dramatic sixfold increase in islet mass (Michael et al.,
2000). These studies prompted the idea that blocking the insulin
receptor with a small molecule or peptide antagonist could
recapitulate these effects, including the induction of β-cell
replication. In fact, treatment with one such insulin receptor
antagonist, a novel peptide named S961, results in
hyperinsulinemia in rats (Schäffer et al., 2003; Schäffer et al., 2008;
Vikram and Jena, 2010).
Furthermore, an additional study of the LIRKO model revealed
that β-cells were specifically induced to replicate in this context
with no effect on α-cells or other non-pancreatic tissues (El
DEVELOPMENT
Development 140 (12)
Ouaamari et al., 2013). This study also demonstrated that the
induced replication could occur in a normal parabiotic partner (with
normal blood glucose and insulin levels) if that animal was joined
with a LIRKO mouse partner, suggesting both that a systemic
factor was at work and that it could work independently of blood
glucose levels. Furthermore, LIRKO-derived serum or a LIRKO
liver explant system was sufficient to recapitulate replication
induction in isolated mouse islets in vitro, although the number of
replicating cells analyzed was very small and the specific factor(s)
responsible were not identified.
Roles for insulin resistance and glucose levels in β-cell
replication
Glucose itself induces β-cell replication in both rodents and
humans, although clearly in the case of diabetes this replication is
insufficient to keep up with demand or with the autoimmune attack
(Bonner-Weir et al., 1989; Porat et al., 2011). Human islets
transplanted into immunodeficient mice can also be induced to
replicate following infusion of glucose (Levitt et al., 2011).
Furthermore, humans with mutations (V91L) in glucokinase that
increase its affinity for glucose similarly show increased β-cell
replication and, as a consequence, larger islets (Kassem et al.,
2010).
Similarly, a high-fat diet induces insulin resistance, which results
in β-cell replication (Terauchi et al., 2007); the total number of βcells in mice on such a diet more than doubled in 20 weeks. The
process of replication under these conditions also requires
glucokinase, as mice haploinsufficient for glucokinase fail to
increase the number of β-cells. By contrast, treatment with
glucokinase activators doubles β-cell replication in both young and
old mice (Stolovich-Rain et al., 2012). Anti-diabetic therapeutics
based on the strategy of glucokinase activation are currently being
investigated, and one beneficial side effect beyond their effects on
hepatic glucose metabolism may be induced β-cell replication
(Matschinsky, 2009).
Recent work in our laboratory extended these studies by
examining the organ-specific transcriptional changes induced by
insulin resistance in order to identify the downstream factors that
enhance β-cell replication. Analysis of hepatocyte transcriptional
changes downstream of S961 revealed upregulation of a novel
hormone, betatrophin (Yi et al., 2013). Remarkably, induction of
betatrophin expression independently of insulin receptor
antagonism increased β-cell replication in mice by more than
tenfold. These data suggest that a novel therapeutic strategy might
be possible for generating more β-cells through increasing the
levels of this newly discovered hormone.
Conclusions and perspectives
In summary, unprecedented progress toward the goal of making
more β-cells has been realized in the last decade. At the same time,
however, additional challenges beyond those discussed above
remain for this field. In particular, we have limited the scope of this
review to the β-cell but acknowledge that addressing the immune
system problem will be essential for treating Type 1 diabetes,
whether through systemic immunomodulation or transplantation of
new β-cells inside an immunoprotective capsule. If new stem cellderived allogeneic β-cells are to be transplanted into patients with
Type 2 diabetes to improve glycemic control, the issue of immune
system rejection will also have to be addressed.
In this regard, a promising strategy to address the immune issue
is the encapsulation of new β-cells in an immunoprotective device
that permits nutrient diffusion but inhibits immune cell infiltration.
Development 140 (12)
One such device has been successfully used for the protected
transplantation of human islets and hESC-derived pancreatic
progenitors into mice (Lee et al., 2009; Xie et al., 2013). An
alternative strategy to avoid allogeneic immune rejection might be
to develop protocols for the differentiation or reprogramming of
patient-specific iPSCs into β-cells, particularly iPSCs that have
been generated by non-viral methods. Early reports suggested that
transplantation of undifferentiated iPSCs into syngeneic mice
resulted in immune attack (Zhao et al., 2011). However, no patientspecific iPSC therapeutic strategy is likely to involve
transplantation of undifferentiated pluripotent stem cells. Two more
recent studies demonstrated that the transplantation of differentiated
cells from syngeneic iPSCs generated no immune reaction or
rejection and that the results of the previous study might be an
artifact of using retrovirally derived iPSCs (Araki et al., 2013;
Guha et al., 2013). Thus, iPSC-derived β-cells have particular
potential to contribute to therapies in the future if scientific
strategies can be developed that overcome the heterogeneity of
differentiation propensities in individual cell lines.
The clinical utility of new sources of β-cells will require
methods to generate homogenous cell populations that lack residual
multipotent cells that could form cysts or tumors. Given the
tremendous progress that has been made towards homogeneity in
the early stages of directed differentiation (to the point of 99%
definitive endoderm at present), this goal should be achievable for
the late stages of differentiation once the appropriate developmental
cues are discovered. In addition, these cell preparations will have
to be generated on a very large scale compared with that typical in
academia, and the first notable progress toward this goal has been
reported recently (Schulz et al., 2012).
In summary, tremendous progress has been made toward both
understanding how β-cells arise and proliferate during normal
development and how functional β-cells might be generated
through novel methods. The challenge of reconstructing a bona fide
β-cell in vitro, whether through stem cell differentiation or
reprogramming, remains open.
Acknowledgements
We thank the members of the D.A.M. laboratory for helpful discussions and
apologize to authors whose studies could not be cited owing to space
limitations.
Funding
Work in the laboratory of D.A.M. is funded by the US National Institutes of
Health; The Leona M. and Harry B. Helmsley Charitable Trust; the Juvenile
Diabetes Research Foundation; and the Howard Hughes Medical Institute.
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
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